Project Summary Abstract Prostate cancer (PCA) is the most frequently diagnosed invasive malignancy in the United States men and is the second leading cause of cancer-related deaths after lung (1). Chemoprevention of PCA has been suggested as a novel and rationale approach to control the growth, malignant progression and metastasis to secondary sites (2-4). In this regard, silibinin, isolated from the seeds of milk thistle (Silybum marianum), has shown promising anti-cancer effects in a number of cell culture and in vivo preclinical models of PCA (4- 9). Silibinin is already consumed extensively in the United States and world-wide as a dietary supplement, and used in clinic as a hepatoprotective agent (10-12). This compound has not shown any toxicity in animal studies as well as in humans (11,12), and recently entered pilot phase II clinical trial in PCA patients after completing a successful phase I clinical trial (13). During the funding period of the current grant, in addition to several other major findings, for the first time we identified that oral silibinin inhibits tumor angiogenesis and epithelial-mesenchymal transition (EMT) as well as distant metastasis in transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model (Cancer Res. August 15, 2008) (14). The present continuation grant builds upon these highly novel observations, and proposes the studies to further establish the efficacy and define the mechanisms for antiangiogenic, antiinvasive and antimetastatic effects of silibinin. We hypothesize that silibinin modulates tumor and tumor microenvironment interaction, and inhibits the processes involved in PCA progression including hypoxia and the hypoxia-driven angiogenesis, invasion, migration and metastasis to distant sites. Together, by targeting these inter-related events, silibinin affords prevention, growth control and therapy of PCA. To test this hypothesis, detailed pre- clinical tumor studies and in depth molecular studies utilizing tumor tissues (and cell culture, where needed) will be performed with silibinin. The specific aims of this competing renewal application are to: 1) examine and define the effect of silibinin on proliferation, metabolism, hypoxia and angiogenesis in PCA; 2) examine and define the inhibitory effect of silibinin on EMT as well as invasive and migratory potential of PCA cells; and 3) identify the anti-metastatic efficacy of silibinin, and define its influence on tumor microenvironment at distant metastatic site. We anticipate that positive outcomes from the proposed studies together with earlier work will further reveal the mechanism-based cancer preventive and therapeutic efficacy of silibinin against PCA. The novelty of the proposed work lies in the fact that for the first time we would be studying in-depth the tumor microenvironment as a target of silibinin to inhibit tumor growth, progression and metastasis, as a strategy for controlling human PCA.